Regulation of Alternative Splicing by the Core Spliceosomal Machinery

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Regulation of alternative splicing by the core spliceosomal machinery

Arneet L. Saltzman,1,2 Qun Pan,1 and Benjamin J. Blencowe1,2,3 1Banting and Best Department of Medical Research, The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada; 2Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP (small nuclear ribonucleoprotein) protein SmB/B9 self-regulates its expression by promoting the inclusion of a highly conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B9 in human cells results in reduced levels of snRNPs and a striking reduction in the inclusion levels of hundreds of additional alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA-binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors. [Keywords: alternative splicing; Sm proteins; snRNP; autoregulation; NMD; exon network] Supplemental material is available for this article. Received October 20, 2010; revised version accepted January 4, 2011.

The production of multiple mRNA variants through indicated that the levels of some of these core splicing alternative splicing (AS) is estimated to take place in components can affect splice site choice. Microarray transcripts from >95% of human multiexon genes (Pan profiling revealed transcript-specific effects on splicing et al. 2008; Wang et al. 2008). AS represents a mechanism in yeast strains harboring mutations in or deletions of for gene regulation and expansion of the proteome. The core splicing components (Clark et al. 2002; Pleiss et al. most widely studied trans-acting factors regulating AS 2007). Knockdown of several core splicing factors in are proteins of the SR (Ser/Arg-rich) and hnRNP (hetero- Drosophila cells resulted in transcript-specific effects on geneous ribonucleoprotein) families, as well as numerous AS reporters (Park et al. 2004). Deficiency of the snRNP tissue-restricted AS factors (for review, see Chen and assembly factor SMN (survival of motor neuron) in a Manley 2009; Nilsen and Graveley 2010). These factors mouse model of spinal muscular atrophy (SMA) resulted generally regulate AS by recognizing cis-acting sequences in tissue-specific perturbations in snRNP levels and in exons or introns and by promoting or suppressing the splicing defects (Gabanella et al. 2007; Zhang et al. 2008). assembly of the spliceosome at adjacent splice sites. In Tiling microarray profiling analysis of fission yeast RNA contrast to these AS regulatory factors, much less is also revealed transcript-specific splicing defects of a tem- known about how or the extent to which components perature degron allele of SMN, and that some of the de- of the basal or ‘‘core’’ splicing machinery modulate splice fects could be alleviated by strengthening the pyrimidine site decisions. tract upstream of the branchpoint (Campion et al. 2010). The spliceosome is a large RNP complex that carries However, the features that underlie the differential sensi- out the removal of introns from pre-mRNAs. It comprises tivity of introns or alternative exons to particular defects the U1, U2, U4/6, and U5 small nuclear RNPs (snRNPs) in the core splicing machinery are not well understood. and several hundred protein factors (for review, see Wahl Many splicing regulatory factors can regulate the AS of et al. 2009). Studies in yeast and metazoan systems have their own pre-mRNAs (autoregulation), and in some cases have been observed to affect the AS of pre-mRNAs encoding other AS factors (for review, see Lareau et al. 2007a; McGlincy and Smith 2008). Such autoregulation 3Corresponding author. E-MAIL [email protected]; FAX (416) 946-5545. and cross-regulation are important for the establishment Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.2004811. and maintenance of AS factor levels across different

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Saltzman et al. tissues and developmental stages. Autoregulation and when NMD is disrupted, as well as when expression of cross-regulation of AS factors can produce protein isoforms SmB is increased exogenously (Saltzman et al. 2008; this with different functional properties (Dredge et al. 2005; study). These results suggested that AS-NMD plays a role Damianov and Black 2010), and have also been shown in in the homeostatic regulation not only of AS regulatory many cases to regulate gene expression by producing factors, but of components of the basal splicing machin- alternative transcripts containing premature termina- ery as well. Moreover, the results further suggested that tion codons (PTCs) that are degraded by nonsense-me- core spliceosomal components may regulate specific AS diated mRNA decay (AS-NMD). Consistent with their events in addition to their well-studied critical roles in functional importance, regulated AS events involved in constitutive splicing. AS-NMD of splicing factors often lie within highly con- In the present study, we investigated the role of the core served or ultraconserved sequence regions (Lareau et al. spliceosomal machinery in AS, focusing on a detailed 2007b; Ni et al. 2007; Yeo et al. 2007; Saltzman et al. investigation of SmB/B9. Our results confirm that SmB/B9 2008). functions in homeostatic autoregulation through the Using AS microarray profiling following knockdown inclusion of a highly conserved PTC-introducing exon of NMD factors, we previously identified highly con- in its own pre-mRNA. This mode of regulation depends served, PTC-introducing alternative exons in genes en- on a suboptimal 59ss associated with the SNRPB PTC- coding multiple core splicing factors (Saltzman et al. introducing exon and appears to be controlled by changes 2008). These genes included SNRPB, which encodes in the level of U1 snRNP as a consequence of SmB/B9 the core snRNP component SmB/B9, a subunit of the depletion. We also show that knockdown of SmB/B9 leads heteroheptameric Sm protein complex that is assem- to a striking reduction in the inclusion levels of many bled by the SMN complex onto the Sm-site of U1, U2, U4, additional alternative exons, which are significantly and U5 snRNAs (for review, see Neuenkirchen et al. enriched in functions related to RNA processing and 2008). The SmB and SmB9 proteins are nearly identical RNA binding. Changes in the inclusion levels of a subset and arise from alternative 39 splice site (ss) usage in the of these alternative exons also appear to control expres- terminal exon of SNRPB (Fig. 1A, top; Supplemental Fig. sion levels of the corresponding mRNAs by AS-NMD. 1), leading to an additional repeat of a short proline-rich Our results thus reveal an important role for the core motif at the C terminus of SmB9 (van Dam et al. 1989). spliceosomal machinery in establishing the inclusion The PTC-introducing alternative exon in SNRPB lies levels of a specific subset of alternative exons, and further within a region of the second intron that is highly suggest that changes in the levels of these alternative conserved in mammalian genomes (Fig. 1A). Splice var- exons control the expression of other RNA processing iants including this PTC-introducing exon accumulate factors.

Figure 1. The inclusion of a highly conserved PTC-introducing alternative exon in SNRPB is affected by SmB/B9 knockdown. (A, top panel) Diagram of the exon/intron structure of the SNRPB gene. The two encoded proteins, SmB and SmB9, arise from alternative 39 ss usage in the final exon. A conservation plot (phyloP), is shown below (generated using the University of California at Santa Cruz genome browser (http://genome.ucsc.edu; Rhead et al. 2010). (Bottom panel) An expanded view of the region between the second and third SNRPB exons. The highly conserved area within this intron contains an alternative exon (‘‘A’’). This alternative exon and its conserved flanking intron regions (boxed in blue) were cloned into a minigene (miniSmB) in which they are flanked by heterologous intron and exon sequences. (B) RT–PCR assays confirm an increase in the level of the PTC-containing SNRPB variant when NMD is abrogated by knockdown of UPF1. See also Supplemental Figures 2 and 3. (C) Knock- down of SmB/B9 leads to more skipping of the SNRPB alternative exon in miniSmB. HeLa cells were transfected with a control nontargeting (NT) siRNA, or an siRNA targeting the 39 untranslated region (UTR) of SmB/B9. Cells were then cotransfected with miniSmB and either an empty vector or a vector encoding a 3xFlag-tagged cDNA, as indicated above the gels. (Top panel) To assay alternative exon inclusion in miniSmB, RT–PCR assays were performed using primers specific for the flanking constitutive exons of the minigene (hatched boxes). Quantifications of the percent exon inclusion level are shown below the gel, and the average percent inclusion 6 standard deviation calculated from at least three independent analyses is shown in the bar graph. The level of SmB/B9 mRNA (middle panel) or 5S rRNA (loading control, bottom panel) was assayed by RT–PCR.

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SmB in alternative splicing regulation

Results creasing SmB/B9 protein levels leads to an increase in the exon-included PTC-containing splice variant (Saltzman Inclusion of a highly conserved PTC-introducing et al. 2008). Together, these results support a role for the alternative exon in SNRPB pre-mRNA is affected highly conserved SNRPB PTC-introducing alternative by levels of core splicing factors exon in the homeostatic autoregulation of SmB/B9 via To initially explore the role of the core spliceosomal AS-NMD. Our results further suggest that components machinery in the regulation of AS, we determined the of the core spliceosomal machinery can function in the effect of SmB/B9 knockdown on the AS of the PTC- regulation of AS, in addition to their well-established introducing SNRPB exon. This exon, together with its roles in constitutive splicing. highly conserved flanking intronic sequences, was cloned into a minigene reporter plasmid containing upstream and Knockdown of the core snRNP protein SmD1 affects downstream heterologous intron and constitutive exon the inclusion of the conserved SNRPB alternative exon sequences (Fig. 1A, miniSmB). Unlike endogenous SmB/B9 To investigate whether other proteins in the Sm hep- transcripts including the PTC-introducing exon (Fig. 1B), tameric complex might also affect the inclusion of the exon-included transcripts derived from the miniSmB re- SNRPB alternative exon, SmD1 was knocked down and porter are not degraded by NMD, as neither the steady- the effect on the AS of miniSmB was assayed by RT–PCR state level nor the half-life of these transcripts is increased (Fig. 2A). As observed for SmB/B9 (Fig. 1C), knockdown of upon disruption of NMD (Supplemental Figs. 2, 3). Mon- SmD1 resulted in more skipping of the miniSmB alterna- itoring transcripts derived from miniSmB therefore allows tive exon. This effect was rescued by exogenous expression an analysis of the splicing regulation of the PTC-introduc- of SmD1, but not of SmB (Fig. 2A). Taken together with the ing SNRPB exon in the absence of effects of NMD. HeLa results described above, these observations suggest that cells were transfected with a control nontargeting (NT) SmB and SmD1 affect AS in a similar but nonredundant siRNA or an siRNA to knock down SmB/B9, followed by manner. One possibility is that depletion of each compo- transfection with the miniSmB reporter plasmid. Knock- nent of the Sm heptameric complex similarly affects the down of SmB/B9 led to increased skipping of the SNRPB overall levels and/or integrity of one or more snRNPs alternative exon in miniSmB (Fig. 1C). Loss of inclusion of in a manner that affects the recognition of the SNRPB the SNRPB alternative exon was rescued by expression of alternative exon. a Flag-epitope-tagged cDNA construct encoding SmB or SmB9 (Fig. 1C). It was also rescued by expression of SmN, Knockdown of Sm proteins affects the levels a tissue-restricted paralog of SmB/B9 that is 93% identical of Sm-class snRNAs to SmB9 (Supplemental Fig. 1). However, loss of inclusion of the SNRPB alternative exon upon SmB/B9 knockdown To investigate how the knockdown of SmB/B9 or SmD1 could not be rescued by expression of the related protein might affect SmB/B9 AS, we next determined whether SmD1, another component of the heteroheptameric Sm reduced levels of either Sm protein affects the steady-state ring (Fig. 1C). Thus, SmB/B9 knockdown leads to an levels of spliceosomal snRNAs using RT–PCR assays. The increase in the exon-skipped miniSmB splice variant (Fig. relative levels of snRNAs measured in total cellular RNA 1C), which represents the protein-coding isoform. Recip- are comparable with those in snRNPs immunoprecipitated rocally, our previous experiments have shown that in- with anti-Sm antibodies (Zhang et al. 2008). This is in

Figure 2. Knockdown of SmD1 leads to more skipping of the SNRPB alternative exon in miniSmB (A), and knockdown of SmB/B9 (B) or SmD1 (C) affects snRNA levels. (A) HeLa cells were transfected with NT or SmD1-specific siRNAs. Cells were then cotransfected with miniSmB and with either an empty vector or a vector encoding the 3xFlag-tagged cDNA indicated above the gel. RT–PCR assays were performed using primers specific for the flanking constitutive exons of the minigene (top panel) or primers specific for SmD1 or b-actin transcripts (bottom panels). The quantifications and bar graph of percent inclusion levels are as in Figure 1. (B) Knockdown of SmB/B9 leads to a decrease in steady-state levels of three of four Sm-class snRNAs that form the major spliceosome (U1, U4, and U5, but not U2) and of the Sm-class snRNAs that form the minor spliceosome (U11, U12, and U4atac). (C) Knockdown of SmD1 shows similar effects, but also a slight decrease in U2 snRNA levels. The levels of LSm (Sm-like)-class snRNAs U6 and U6atac were not decreased in either the SmD1 or SmB/B9 knockdown.

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Saltzman et al. agreement with previous studies indicating that the pool motifs either act through different trans-acting factors, or of ‘‘Sm-free’’ snRNAs is relatively small (Sauterer et al. are involved in regulation in a manner that is redundant 1988; Zieve et al. 1988). Consistent with an important with other sequence motifs. However, in several cases, the role for Sm core assembly in snRNP stability (Jones and mutation and/or deletion of a particular sequence reduced Guthrie 1990), knockdown of SmB/B9 led to a decrease in or eliminated the impact of SmB/B9 knockdown on in- Sm-class snRNAs (U1, U4, U5, U11, U12, and U4atac), clusion of the alternative exon. In particular, a deletion with the exception of U2 (Fig. 2B). Knockdown of SmD1 from the seventh to the 18th nucleotide downstream from also led to a decrease in Sm-class snRNAs, including the 59ss led to an increase in the inclusion of the alternative a slight decrease in U2 (Fig. 2C). The similar effects exon. However, in contrast to wild-type miniSmB, knock- observed for both knockdowns are consistent with a down of SmB/B9 had no detectable effect on the exon shared role for Sm proteins in the inclusion of alterna- inclusion level of this mutant reporter (Supplemental Figs. tive exons through modulating the overall levels of one or 4, 5). Substitution of the same sequence with the 12-base more snRNPs. linker also led to an increase in exon inclusion, but, in contrast to the deletion mutant, did not prevent increased Cis-acting elements regulating inclusion of the SNRPB exon skipping caused by SmB/B9 knockdown (Supplemen- alternative exon tal Figs. 4, 5). Examination of sequences adjacent to the 59ss To investigate the mechanism by which knockdown of Sm created by this pair of mutants revealed that the deletion proteins and the associated reductions in snRNP levels but not the substitution created a sequence with increased affect AS of SmB/B9 pre-mRNA, we performed a detailed potential for base-pairing to U1 snRNA. These results mutagenesis analysis of the miniSmB reporter. Recapitula- therefore suggest that regulation of the SNRPB alternative tion in this reporter (Fig. 1) of the Sm protein-dependent AS exon by Sm protein levels depends on sequences at or prox- effects seen for endogenous transcripts (Saltzman et al. imal to the exon 59ss. Therefore, the role of 59ss sequences 2008) suggested that all of the cis-acting elements required in regulating the inclusion of the SNRPB alternative exon for mediating regulation are contained within the highly was investigated in greater detail. conserved SNRPB PTC-introducing exon and/or its flank- Mutations that strengthen the 59ss reduce the effects ing intronic sequences. Linker-scanning mutagenesis was of Sm protein knockdown performed, in which successive 12-base segments of the alternative exon and its upstream and downstream flank- To determine the role of 59ss sequences in Sm protein ing introns were deleted or substituted with a linker se- knockdown-dependent effects on miniSmB AS, 59ss muta- quence. This strategy identified sequences in the exon and tions were introduced into miniSmB, and the level of exon introns acting as splicing enhancers or silencers. These inclusion was assayed in control and SmB/B9 knockdown elements are concentrated near splice sites (Supplemental conditions (Fig. 3A). Two mutations that strengthen the Fig. 4A). In most cases, knockdown of SmB/B9 ledtoacom- 59ss increase its inclusion level, yet, unlike wild-type parable increase in exon skipping of the mutated or deleted miniSmB but similar to the deletion mutant described minigenes, as observed for the wild-type miniSmB (Supple- above that strengthens the 59ss, show very little skipping mental Fig. 4B). These results suggest that these sequence when SmB/B9 is knocked down (Fig. 3A, consensus and

Figure 3. Mutations that strengthen the 59ss, but not mutations that strengthen the 39ss, reduce the effects of SmB/B9 knockdown on miniSmB AS. HeLa cells were transfected with NT or SmB/B9-specific siRNAs. Cells were then cotransfected with wild-type (wt) miniSmB or with minigenes harboring mutations in the 59ss (A)or39ss (B) as indicated above the gel images, along with either empty vector or a vector encoding the 3xFlag-tagged SmB cDNA construct. (A) Increasing the 59ss strength (consensus and strong) results in increased percent inclusion relative to wild type, as well as a marked reduction in the effect of SmB/B9 knockdown on percent inclusion. Secondary mutations introduced into the ‘‘consensus’’ construct to decrease the 59ss strength (U+6C and A+3G) restore sensitivity to SmB/B9 knockdown. (B) Increasing the 39ss strength results in increased percent inclusion relative to wild type, but no reduction in the effect of SmB/B9 knockdown. RT–PCR assays and quantifications of the percent exon inclusion are as in Figure 1C. (c) Pseudouridine.

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SmB in alternative splicing regulation strong). However, SmB/B9 knockdown sensitivity is re- stored by the introduction of additional mutations in the 59ss consensus minigene that weaken the 59ss (U+6C, A+3G) (Fig. 3A). In contrast, a mutation that strengthens the 39ss (U10ACAGjG) also increases the inclusion level of the minigene, yet does not abrogate the effect of SmB/B9 knockdown (Fig. 3B). Similar results were obtained for two other mutations that increase the strength of the 39ss (Supplemental Fig. 6). Thus, minigenes with a strong 59ss or 39ss have similarly high basal levels of exon inclusion (;90%), yet the effect of SmB/B9 knockdown on exon skipping is only abrogated for minigenes that have mutations that strengthen the 59ss. Taken together with the observation that SmB/B9 or SmD1 knockdown results in reduced levels of U1 snRNP (Fig. 2), our data provide evidence that the suboptimal 59ss of the SNRPB PTC- introducing alternative exon is necessary for its sensitivity to Sm protein depletion.

A widespread role for core splicing factors in promoting the inclusion of alternative exons To determine whether reducing the levels of SmB/B9,and thus Sm-class snRNPs, via SmB/B9 knockdown affects the inclusion of alternative exons from other genes, high- throughput RNA sequencing (RNA-seq) was performed Figure 4. Quantitative analysis of AS by RNA-seq reveals that knockdown of SmB/B9 leads to increased skipping of alternative on RNA from HeLa cells following knockdown of SmB/B9 exons. (A) Western blots indicate that SmB/B9 (top panels) or using an siRNA pool, and on RNA from cells transfected SRSF1 (bottom panels; also known as SF2, ASF, SFRS1) were with an NT siRNA pool as a control (Fig. 4A). To compare efficiently depleted by siRNA transfections. (NT) NT siRNA. and assess the specificity and extent of the effects of SmB/ Serial dilutions of protein extract indicate that the blots are B9 knockdown on AS with those of a relatively well-defined semiquantitative. (B) The percentage of alternative exons (left) splicing regulator, we used another siRNA pool to knock or constitutive exons (right and inset) showing changes in down the SR family protein SRSF1 (also known as ASF, inclusion levels upon knockdown of SmB/B9 or SRSF1 when SF2, SFRS1) (Fig. 4A). A comparable knockdown efficiency compared with inclusion levels in cells transfected with a con- of >85% was achieved in both factor knockdowns. trol NT siRNA are shown in a bar graph. (C) Distinct effects of The RNA-seq reads (50 nucleotides [nt]) were mapped to SmB/B9 and SRSF1 knockdowns. The percent exon inclusion values in the control NT, SmB/B9, and SRSF1 knockdowns are exon–exon junctions in a database of EST/cDNA-sup- shown for 268 alternative exons found to have a $30% in- ported cassette-type AS events (see the Materials and clusion change (increased skipping) in the SmB/B9 knockdown Methods). Counts of reads mapping to included versus when compared with the control (NT). The AS events are skipped exon junctions were used to calculate the percent ordered from top to bottom by the absolute difference in percent inclusion level of these alternative exons. Alternative inclusion between the SRSF1 knockdown and the control (NT). exons meeting filtering criteria based on junction read coverage (n = 5752) (Supplemental Table 1) were analyzed, and the proportion of cassette alternative exons changing strongly affected by SmB/B9 knockdown were not similarly in inclusion level between each knockdown and the affected by knockdown of SRSF1, as shown in the heat map control was plotted (Fig. 4B, left). of the percent inclusion of alternative exons showing Knockdown of SmB/B9 specifically reduced the inclusion $30% change in the SmB/B9 knockdown (Fig. 4C). Thus, levels of a large number of alternative exons, and, overall, the SmB/B9 and SRSF1 knockdowns affected distinct sets affected the inclusion levels of more than twice the of alternative exons, and nearly all changes in alternative number of alternative exons than were affected by knock- exon inclusion following knockdown of SmB/B9 represent down of SRSF1. Relative to the control knockdown, 18% increased exon skipping. (n = 1035) of alternative exons were $10% more skipped in To determine the effect of knockdown of SmB/B9 on the the SmB/B9 knockdown, whereas only 0.8% (n = 48) were inclusion levels of constitutive exons, the RNA-seq reads $10% more included. Moreover, all alternative exons were aligned to exon–exon junctions in a database of high- showing a change in percent inclusion of $30% (n = 268) confidence internal constitutive exons using the same were more skipped in the SmB/B9 knockdown compared filtering criteria as applied for alternative exons (refer to with the control. In contrast, knockdown of SRSF1 resulted the Materials and Methods; Supplemental Table 2). Only in 7.4% (n = 423) of alternative exons changing by $10% 1.9% of the constitutive exons (160 of 8626) showed $10% inclusion, and 61% of these were more skipped while 39% skipping when SmB/B9 was knocked down, compared with were more included (Fig. 4B, left). Most alternative exons 18% (1035 of 5752) of alternative exons (P < 1 3 104; x2)

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Saltzman et al.

(Fig. 4B). These results indicate that a specific subset of Consistent with previous results (Stamm et al. 2000; alternative exons is particularly sensitive to reduced Clark and Thanaraj 2002; Itoh et al. 2004), the 39ss (Fig. 6A, snRNP levels as a consequence of SmB/B9 knockdown, top panel) and 59ss (Fig. 6A, middle panel) of the profiled whereas constitutive exon inclusion is largely unaffected. alternative exons are, on average, weaker than those of the To assess the accuracy of alternative exon inclusion constitutive exons (39ss: P = 4.5 3 1027;59ss: P = 5.7 3 levels and knockdown-dependent changes detected by 1041, Wilcoxon rank sum test) (Fig. 6A, see legend, analysis of the RNA-seq data, AS events analyzed above bottom panel). However, the average strength of the 39ss (n = 5752) were divided into three equally sized groups of alternative exons whose inclusion is affected by the based on their junction read coverage. Events showing SmB/B9 knockdown is higher than that of the other more skipping, no change, or more inclusion when com- profiled alternative exons (8.57 vs. 8.14; P = 0.02, Wilcoxon paring the SmB/B9 knockdown with the control were rank sum test), and is not significantly different from the selected from these three groups, and the alternative exon average strength of the 39ss of the constitutive exons (8.57 inclusion levels were measured by RT–PCR using primer vs. 8.60) (Fig. 6A). In contrast, the average strength of the pairs targeting the flanking constitutive exons (n = 28 59ss of alternative exons affected by SmB/B9 knockdown events). The RNA-seq measurements for percent inclu- was lower than that of the other profiled alternative exons, sion levels agreed very well with those from the RT–PCR although this difference was not statistically significant data (r = 0.97) (Fig. 5A). Representative RT–PCR results (8.06 vs. 8.34) (Fig. 6A). In addition, alternative exons are shown in Figure 5B, and all results are shown in affected by SmB/B9 knockdown were, on average, shorter Supplemental Figure 7. Twenty-one of these AS events (median = 86 nt) than the other alternative exons (median = were also assayed in two additional independent knock- 104 nt; P = 3.9 3 1011,Wilcoxonranksumtest)(Fig.6B). downs of SmB/B9, and the knockdown-dependent AS Thus, alternative exons showing more skipping when changes were confirmed in all cases (Supplemental Fig. 8). SmB/B9 is knocked down are, on average, shorter and have a stronger 39ss than other profiled alternative exons. These Characteristics of SmB/B9 knockdown-dependent results are consistent with our SNRPB minigene mutagen- alternative exons esis results, in which the effect of SmB/B9 knockdown on exon inclusion was not reduced by mutations increasing The results from analyzing the miniSmB reporter mutants the 39ss strength, but was essentially eliminated by muta- indicated that the presence of a suboptimal 59ss is an tions increasing the 59ss strength (Fig. 3). important determinant of the effect of SmB/B9 knockdown on alternative exon inclusion levels. To address whether Changes in transcript levels associated with this and/or other sequence features account more gener- SmB/B9 knockdown-dependent PTC-introducing ally for the effects of SmB/B9 knockdown on exon in- alternative exons clusion levels, we next investigated the relationship between splice site strength and sensitivity to SmB/B9 To investigate the functional consequences of SmB/B9 knockdown by comparing the average splice site strength knockdown-dependent AS changes, the capacity of these scores (Yeo and Burge 2004) of the affected alternative AS events to produce NMD-targeted isoforms that affect exons with those of other alternative and constitutive overall mRNA expression levels of the corresponding exons analyzed above by RNA-seq. genes was next determined. The mRNA expression levels

Figure 5. Changes in alternative exon inclusion levels measured by RNA-seq are confirmed by RT–PCR assays. (A) Scatter plot showing agreement between percent inclusion of 27 alternative exons in the three knockdowns as measured by RT–PCR versus RNA-seq (left), and between differences in inclusion levels (knockdown relative to control NT) for the same 27 alternative exons (right). (B) Representative RT–PCR assays using primers annealing to flanking constitutive exons. For all RT–PCR assays, see Supplemental Figure 7. Gene names: (hnRNPAB) hnRNPA/B; (hnRNPH1) hnRNPH1; (SRSF7; also known as SFRS7, 9G8) serine/ arginine-rich splicing factor 7; (SFRS18; also known as SRrp130) splicing factor, arginine/serine-rich 18; (DDX11) DEAD/H (Asp–Glu–Ala–Asp/His)-box polypeptide 11; (DDX49) DEAD-box polypeptide 49; (CPSF7) cleavage and polyadenylation-specific factor 7, 59 kDa; (CENPN) centromere protein N. The number following the period designates the AS event ID. (Names with an asterisk) PTC upon skipping.

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SmB in alternative splicing regulation

Figure 6. Characteristics of alternative exons affected by knockdown of SmB/B9.(A,B) Cumu- lative distribution function (CDF) plots of 39ss scores (A, top), 59ss scores (A, middle), and exon lengths (B) for alternative and constitutive exons profiled by RNA-seq (Fig. 4). Alternative exons that show a pronounced increase in skipping ($30%) upon knockdown of SmB/B9 are plotted separately from other profiled alternative exons, as showninthebottom panel of A.(C) CDF of the fold change in overall mRNA transcript level

(log2 scale; SmB/B9 knockdown vs. control) of transcripts containing AS events that are more skipped upon knockdown of SmB/B9 compared with the control knockdown. Transcripts containing AS events that introduce a PTC upon exon inclusion or skipping or that do not introduce a PTC (No PTC) are plotted separately, as shown in the legend below the plot. (alt) Alternative; (const) constitutive. for genes containing SmB/B9 knockdown-sensitive alter- levels of these genes in response to reduced snRNP native exons ($10% more skipping) were measured by levels as a consequence of SmB/B9 depletion. aligning RNA-seq reads to RefSeq transcripts (see the Materials and Methods). The fold change in expression SmB/B9 knockdown affects AS events in RNA level in the SmB/B9 knockdown compared with the processing factor genes control was plotted for AS events that do not introduce a PTC, and for events that introduce a PTC in the exon- The functional categories represented in genes containing included or exon-skipped isoform (Fig. 6C). For exons SmB/B9 knockdown-sensitive alternative exons were ex- more skipped in the SmB/B9 knockdown that introduce amined using Gene Ontology (GO) term enrichment (Fig. a PTC upon skipping, the overall mRNA levels from the 7A; Supplemental Table 3). Genes containing alternative genes were, on average, reduced in the SmB/B9 knock- exons showing more skipping upon knockdown of SmB/ down, and the median fold change was significantly B9 ($30%) were significantly enriched for terms related to different from that of non-PTC-introducing events nucleic acid binding and RNA processing (Fig. 7A). These (P = 2.5 3 108, Wilcoxon rank sum test) (Fig. 6C). genes include spliceosome components; splicing regula- Examples of three such AS events are shown in Figure 5B tory factors such as SR, SR-related, and hnRNP family (DDX49.6, DDX11.11, and CPSF7.4). Conversely, for proteins; mRNA 39-end processing factors; RNA heli- exons more skipped in the SmB/B9 knockdown that cases; and other RNA-binding proteins (e.g., Fig. 5B; Sup- introduce a PTC upon inclusion, the overall mRNA plemental Figs. 7,8). Similar results were also obtained levels from the corresponding genes were, on average, using Pathway Commons annotations (Cerami et al. higher in the SmB/B9 knockdown (P = 4 3 103) (Fig. 6C). 2011), which are compiled mostly from protein–protein These results are consistent with AS-NMD acting to interaction data (see the Materials and Methods; Supple- both positively and negatively modulate transcript mental Table 3). These results therefore support the

Figure 7. Model for the role of core snRNP proteins in AS regulation. (A) Enriched GO terms (P < 0.005 and FDR < 0.1) annotating genes containing exons affected by SmB/B9 knockdown ($30% more skipping) are represented as a network of gene sets. Each node represents the set of genes annotated with the indicated GO term. Node size is proportional to the number of genes annotated by the term (indicated by the node label), and edge thickness is proportional to the number of genes in common between the sets. (B)Modelsumma- rizing our data. Levels of SmB/B9 are regulated through homeostatic feedback acting via AS- NMD. The related tissue-restricted Sm protein SmN also cross-regulates SmB/B9 (see the Discus- sion). SmB/B9 levels, through effects on functional snRNP concentrations, also promote inclusion of alternative exons in many other genes, a subset of which also regulate transcript levels via NMD.

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Saltzman et al. conclusion that an important role for alternative exons Our global analysis of AS events displaying altered in- affected by changes in the level of the core spliceosomal clusion upon knockdown of SmB/B9 and the associated snRNP machinery is to coordinately control the expres- depletion of snRNPs also revealed an association of these sion of many RNA processing factors and other regulators alternative exons with relatively weak 59ss and with 39ss of RNA. that were, on average, as strong as those of constitutive exons. Thus, the reduced inclusion levels of a large number Discussion of alternative exons as a consequence of Sm protein depletion may be mediated more generally by reduced rates In this study, we identify a network of alternative exons of interaction between the 59ss and U1 snRNP. These in RNA processing factor genes that is controlled by the findings may relate to previous observations revealing that levels of the core spliceosomal machinery. We provide the Sm complex contributes to the stability of the U1 insight into how these exons are regulated, and their roles snRNA:pre-mRNA interaction in yeast (Zhang et al. 2001), in both feedback and coordinated control of gene expres- and that proper Sm core assembly is essential for snRNP sion (summarized in Fig. 7B). stability (Jones and Guthrie 1990; Zhang et al. 2008). Our results also relate to in vitro studies demonstrating that differential binding of U1 snRNP to stronger or weaker 59ss AS regulation by general splicing factors can affect the inclusion levels of a reporter alternative exon Previous studies have shown that mutation, deletion, or (Kuo et al. 1991). Furthermore, our data support evidence knockdown of core spliceosomal and spliceosome assem- that altering the kinetics of spliceosomal rearrangements bly factors can result in altered splicing patterns in yeast can affect splice site selection (Query and Konarska 2004; (Clark et al. 2002; Pleiss et al. 2007; Kawashima et al. 2009; Yu et al. 2008). Such kinetic competition between splice Campion et al. 2010), fly (Park et al. 2004), and mammalian sites may provide a basis for the changes in the alternative cells (Massiello et al. 2006; Pacheco et al. 2006; Hastings exon inclusion levels that we observe in this study, where et al. 2007; Zhang et al. 2008; Baumer et al. 2009). In specific splice sites are no longer efficiently recognized addition, supporting our finding that knockdown of SmB/ when core splicing components, normally present at B9 and associated snRNP components affects AS levels, saturating levels, may become rate-limiting (Smith et al. a recent genome-wide siRNA screen implicated SmB/B9 in 2008; Graveley 2009; Nilsen and Graveley 2010). the AS of two Bcl2 family apoptosis factors (Moore et al. 2010). Thus, as is well established for splicing regulatory A network of coregulated AS events in RNA processing factors, it is emerging that the relative concentration or factor genes activity of general splicing factors can affect splice site selection. Consistent with an important physiological role Our finding that alternative exons affected by SmB/B9 for the core spliceosomal machinery in the regulation of knockdown are significantly enriched in genes encoding AS, components of snRNPs are differentially expressed in RNA processing/binding factors, and that some of these AS mammalian cells and tissues (Grosso et al. 2008; Castle events regulate transcript levels via AS-NMD, suggest an et al. 2010), and several core splicing factors are differen- important role for the core spliceosomal machinery in the tially expressed during development and in tissues of the regulation of AS events that likely function to maintain fly (Park et al. 2004). Evidence for critical physiological coordinated expression levels of many RNA processing fac- regulatory roles for core spliceosomal components and tors. These results extend previous observations of feedback assembly factors have also emerged from the study of and cross-regulation among specific splicing factors via AS- certain human diseases. For example, mutations in com- NMD. For example, in addition to several reported exam- ponents of the U4/U6.U5 tri-snRNP particle are associated ples of feedback regulation of splicing components and with retinitis pigmentosa (for review, see Mordes et al. other RNA-binding proteins (for review, see Lareau et al. 2006), mutations in the U4/U6 snRNP recycling factor 2007a; McGlincy and Smith 2008), cross-regulation through SART3 (also known as p110) are associated with the skin AS-NMD has been found to occur between gene family disorder disseminated superficial actinic porokeratosis members and paralogs of auxiliary splicing regulators, (ZH Zhang et al. 2005), and loss or mutation of the widely including HNRNPL and HNRPLL (Rossbach et al. 2009); expressed snRNP assembly factor SMN1 causes SMA (see PTBP1 , PTBP2 (also known as nPTB/brPTB), and ROD1 above; for review, see Burghes and Beattie 2009). Although (also known as PTBP3) (Wollerton et al. 2004; Boutz et al. the specific mechanisms and transcript targets that are 2007; Makeyev et al. 2007; Spellman et al. 2007); the fly responsible for these diseases are largely unknown, these homologs of the SR protein SRSF3 (also known as SRp20), studies point to the importance of maintaining appropriate Rbp1 and Rbp1-like (Kumar and Lopez 2005); the T-cell- expression of the core splicing machinery. restricted intracellular antigen-1 RNA-binding proteins Through detailed mutagenesis of sequences surrounding TIA1 and TIAL1 (also known as TIAR) (Le Guiner et al. a highly conserved PTC-introducing exon in the SNRPB 2001; Izquierdo and Valcarcel 2007); CUGBP and ETR3- gene, and the observation that knockdown of SmB/B9 like family members CELF1 (also known as CUGBP1) results in reduced levels of U1 but not U2 snRNP, our and CELF2 (also known as CUGBP2)(Dembowskiand results demonstrate that the strength of the 59ss of an Grabowski 2009); and the muscleblind-like factors MBNL1 alternative exon is an important determinant of its sensi- and MBNL2 (Lin et al. 2006; Kalsotra et al. 2008). Interest- tivity to depletion of this core spliceosomal component. ingly, analogous to the observations in the present study,

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SmB in alternative splicing regulation it was shown recently that the minor spliceosomal snRNP knockdown/minigene experiments, cells were transfected with components U11-48K and U11/U12-65K (also known as siRNAs and then cotransfected with minigene plasmids and SNRNP48 and RNPC3, respectively) are regulated post- cDNA expression plasmids 2 d later. Cells were then harvested transcriptionally through a feedback mechanism involv- 2 d after the plasmid transfection. For RNA-seq experiments, cells ing AS and AS-NMD (Verbeeren et al. 2010). were harvested 3 d post-siRNA transfection. Treatment with cycloheximide and mRNA half-life experiments are described in In addition to the examples summarized above, we the legends for Supplemental Figures 2 and 3. propose that there is cross-regulation between SmB/B9 and its closely related paralog, SmN, encoded by the imprinted SNRPN locus that arose by duplication of the RNA and protein isolation, RT–PCR, and Western blotting SNRPB gene in mammals (Rapkins et al. 2006). Unlike Total RNA was isolated using TRI reagent (Sigma) according to the SmB/B9, which is widely expressed, SmN is expressed manufacturer’s instructions. RT–PCR assays were performed primarily in the brain and heart (Supplemental Fig. 1; using 5–10 ng of input total RNA in a 10-mL reaction using the McAllister et al. 1988, 1989). The expression of SmN is One-Step RT–PCR kit (Qiagen) with or without addition of 32 often disrupted in Prader-Willi syndrome (PWS), a disorder a- P-dCTP (Perkin-Elmer). Total protein lysates in radioimmu- with a range of symptoms, including cognitive impairment noprecipitation buffer supplemented with complete mini-EDTA- free protease inhibitor cocktail (Roche) were separated by SDS- (for review, see Cassidy and Driscoll 2009). However, in PAGE, and Western blotting was performed with the monoclonal brain tissue from PWS individuals or mouse models lack- antibody Y12 to detect SmB/B9 (Lerner et al. 1981), with mAb96 ing SmN expression, SmB expression is up-regulated to detect SRSF1 (Hanamura et al. 1998), or with anti-a-tubulin through a previously unknown mechanism (Yang et al. (Sigma, T6074). Primers for RT–PCR analysis of snRNAs were 1998; Gray et al. 1999a,b). Our results strongly suggest that as described (Zhang et al. 2008). Primer sequences for AS events this apparent dosage compensation occurs by cross-regula- are available on request. tion between SmN and SmB/B9 involving the highly conserved PTC-introducing exon we defined in SNRPB. Plasmid construction In particular, we observed that SmN, SmB9,andSmB SNRPB expressed from cDNAs display very similar activity in To construct miniSmB, the 124-nt PTC-introducing alternative exon along with 124 nt of upstream intron and 122 the restoration of inclusion levels of the SNRPB PTC exon nt of downstream intron was amplified by PCR from HeLa when endogenous SmB/B9 is knocked down. It follows, genomic DNA and cloned into the XhoI/NotI sites of the therefore, that elevated SmN expression in the brain would pET01/Exontrap vector (Mobitec). The amplified SNRPB fragment lead to reduced levels of SmB/B9 by promoting inclusion of corresponds to human chr20:2395715-2396221 (Hg18; reverse its PTC exon. This repression would be relieved when strand). Mutations of miniSmB were introduced by site-directed SmN expression is disrupted in PWS, allowing increased mutagenesis or by overlap PCR using Phusion polymerase (NEB). expression of SmB/B9. Such a mechanism could also relate For expression of 3xFlag-tagged proteins, the cDNAs encoding to the concomitant reduction in SmB/B9 expression upon SmB, SmD1, or SmN from the human ORFeome collection (Open increased expression of SmN in the postnatal relative to the Biosystems) were cloned into pMT3989 (a gift from Marcia Roy, embryonic rodent brain (Grimaldi et al. 1993). Our results Tyers Laboratory) using Gateway LR Clonase II (Invitrogen). The SmB9 construct was generated by site-directed mutagenesis of show that these highly similar proteins have overlapping SmB. All constructs were verified by sequencing. functions, and therefore are capable of cross-regulation via AS-NMD in vivo. It is also interesting to consider that cross-regulation of these paralogs via AS-NMD may reduce Analysis of AS and transcript levels by RNA-seq the phenotypic severity of loss of SmN expression. Total RNA was submitted to Illumina for the FastTrack mRNA- In summary, we uncovered a large set of alternative seq service, and 50-nt reads were generated (siNT, 4923MB; exons that are controlled by levels of the core spliceosomal siSmB/B9 2551 MB; siSRSF1: 2814 MB). Cassette AS events (n = machinery. Some of these exons affect mRNA levels by 27,240) were mined by aligning EST/cDNA sequences to the introducing PTCs that elicit NMD. A subset of the affected genome essentially as described (Pan et al. 2004, 2005). The mRNA-seq reads were mapped to exon–exon junction sequences exons likely plays a critical role in maintaining balanced in this database of cassette AS events as described (Pan et al. 2008). levels of splicing and other RNA-associated factors. These Exon–exon junctions were filtered for coverage in all three results thus provide new insight into regulated exon samples by matching to one or both of the following two criteria, networks as well as the functions of core spliceosomal where exonA is the alternative exon, and exons C1 and C2 are the components. upstream and downstream flanking exons, respectively: (1) $20 reads matching the skipped junction (exonC1:exonC2), or (2) $20 Materials and methods reads matching the included junction with higher coverage and $15 reads matching the included junction with lower coverage (included junctions: exonC1:exonA and exonA:exonC2). Percent Cell culture, siRNA, and plasmid transfection inclusion was calculated using the junction read counts as follows: HeLa cells were grown in DMEM (Sigma) supplemented with 10% avg(C1:A,A:C2)/[(C1:C2 + avg(C1:A,A:C2)]. In parallel, sequencing fetal bovine serum (Sigma) and penicillin/streptomycin (Gibco). reads were also aligned to RefSeq transcripts, and transcript levels For knockdowns, cells were transfected with siRNAs (On-Target- were estimated using the reads per kilobase of exon per million Plus-modified, Dharmacon) at a final concentration of 100 nM mapped reads (RPKM) calculation (Mortazavi et al. 2008). Data for using Dharmafect1 (Dharmacon) according to the manufacturer’s filtered AS events (n = 5752) are provided in Supplemental Table 1. instructions. Plasmids were transfected using Lipofectamine2000 Sequencing read data were deposited in the NCBI Gene Expression (Invitrogen) according to the manufacturer’s instructions. For Omnibus (accession number GSE26463).

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A database of internal consecutive constitutive exon triplets References (n = 33,319) was constructed using the Galaxy tool (http:// main.g2.bx.psu.edu; Blankenberg et al. 2007; Blankenberg et al. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry 2010) as follows: Exons from University of California at Santa JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. 2000. Cruz (UCSC) known genes that overlapped with genes in our AS Gene Ontology: Tool for the unification of biology. Nat database were selected following removal of exons that overlap Genet 25: 25–29. sequences in the UCSC knownAlt track as well as removal of Baumer D, Lee S, Nicholson G, Davies JL, Parkinson NJ, Murray exons that overlap our cassette AS database. Reads were aligned LM, Gillingwater TH, Ansorge O, Davies KE, Talbot K. 2009. to the exon–exon junctions and filtered as described above for the Alternative splicing events are a late feature of pathology in AS events. 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Nucleic To compare the frequency of SmB/B9 knockdown-dependent Acids Res 31: D685–D690. doi: 10.1093/nar/gkq1039. changes in percent inclusion of alternative versus constitutive Chen M, Manley JL. 2009. Mechanisms of alternative splicing exons profiled by RNA-seq, the x2 test was used. Sample sizes are regulation: Insights from molecular and genomics ap- given in the text. To compare the median splice site strengths, proaches. Nat Rev Mol Cell Biol 10: 741–754. the lengths of profiled exons, and the changes in transcript levels Clark F, Thanaraj TA. 2002. Categorization and characterization of between subsets of AS events, the nonparametric Wilcoxon rank transcript-confirmed constitutively and alternatively spliced sum test was used. Sample sizes are shown in Figure 6. introns and exons from human. Hum Mol Genet 11: 451–464. Clark TA, Sugnet CW, Ares M Jr. 2002. Genomewide analysis of Acknowledgments mRNA processing in yeast using splicing-specific micro- arrays. Science 296: 907–910. We thank John Calarco for helpful comments on the manuscript. Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, We thank Drs. Adrian Krainer, Timothy Hughes, and Mike Tyers Workman C, Christmas R, Avila-Campilo I, Creech M, Gross for reagents. This work was supported by grants from the B, et al. 2007. Integration of biological networks and gene Canadian Cancer Society (formerly National Cancer Institute of expression data using Cytoscape. Nat Protoc 2: 2366–2382. Canada) and Canadian Institutes of Health Research (MOP- Damianov A, Black DL. 2010. Autoregulation of Fox protein 67011) to B.J.B., and in part by a grant to B.J.B. and others from expression to produce dominant negative splicing factors. Genome Canada funded through the Ontario Genomics Insti- RNA 16: 405–416. tute. A.L.S. performed the experiments and analyzed the data. Dembowski JA, Grabowski PJ. 2009. The CUGBP2 splicing Q.P. performed RNA-Seq read alignments and assisted with data factor regulates an ensemble of branchpoints from perimeter analysis. A.L.S. and B.J.B. designed the experiments and wrote binding sites with implications for autoregulation. PLoS the manuscript. Genet 5: e1000595. doi: 10.1371/journal.pgen.1000595.

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384 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press

Regulation of alternative splicing by the core spliceosomal machinery

Arneet L. Saltzman, Qun Pan and Benjamin J. Blencowe

Genes Dev. 2011, 25: Access the most recent version at doi:10.1101/gad.2004811

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